System for combined electric, magnetic, and convective acceleration of chemical and biochemical reactions and methods of use thereof

ABSTRACT

The invention relates to a microfluidic system based on active control of flow resistance and balancing pressures in microfluidic channels and an improved method for enhancing reactions with magnetic beads used in disposable microfluidic devices and cartridges for use in, but not limited to, in-vitro diagnostics. The microfluidic system and device of the invention does not utilize mechanical moving parts to control the fluid flow and has no external fluidic connection to the instrument or fluidics controller. The microfluidic system and device combines magnetic control over the movement of magnetic detection beads with electric field and convective enhancement of the movement of analytes and/or or reagentss surrounding the magnetic detection beads, thereby enabling movement of magnetic beads and analytes in the same direction or in different directions. The present invention thereby provides significantly enhanced interactions between analytes and/or reagents with the magnetic beads, which yields higher sensitivity for detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a PCT International Patent Application that claims priority to U.S. Provisional Patent Application No. 63/119,362, filed on Nov. 30, 2020, and U.S. Provisional Patent Application No. 63/119,421, filed on Nov. 30, 2020, the entire disclosures of which are incorporated herein by reference.

GOVERNMENT INTEREST

This invention was made with government support under 5 R44 HD084019-03 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to controlling, accelerating, and performing chemical and biochemical reactions in microfluidics systems by combining electric field, magnetic field, and convective acceleration of transport of molecules in analytical systems.

BACKGROUND OF THE INVENTION

Various apparatuses and methods for controlling and accelerating chemical or biochemical reactions in microfluidics devices are known in the art. Electrokinetic phenomena utilizing electric field-controlled transport, including electroosmosis, electrophoresis, or dielectrophoresis are used to control the chemical or biomolecular transport of molecules in the analytic devices in the sample preparation or detection steps. Magnetic field acceleration of reactions, for instance in magnetic bead-based sample preparation methods, for extraction of the analyte of interest on the surface of the beads and separation from a debris of a clinical sample are well known techniques. Molecular biology reactions, primarily those based on hybridization, polymerization or amplification of the analyte are generally known to be slow processes, controlled by slow diffusion of reagent molecules and analyte molecules. Often hybridization reactions, e.g., between oligonucleotide probes and DNA or RNA targets as well as between protein analytes and antibody captures may require hours to overnight incubation times. Polymerization reactions, for amplification of DNA or RNA target specific to a particular gene or pathogen of interest typically require 2 hours or more to complete. For instance, in the polymerase chain reactions (PCR) (Taylor G. R. “Polymerase chain reaction: basic principles and automation” in “PCR: a Practical approach”. edited by Mcpherson M J et al., Oxford Univ. Press 1991), real-time PCR (Higuchi et al., “Kinetic PCR analysis: real-time monitoring of DNA amplification reactions”. Bio/Technology 1 1:1026-1030 (1993), 1026-1030), enzymatic reactions of ligation, displacement, or similar, the rate determining step is always slow diffusion of the analyte molecule and specific detection capturing probes or the accessibility of the analyte to the detection surface. The same is the case in novel next generation sequencing (NGS) methods (Barton E. Slatko, Andrew F. Gardner, and Frederick M. Ausubel, Overview of Next Generation Sequencing Technologies, Curr Protoc Mol Biol. 2018 April; 122(1); U.S. patent application Ser. No. 10,704,091 B2, Genotyping by Next Generation Sequencing) where multiple and repeated polymerization, hybridization and de-hybridization or denaturing reactions are performed during one long analysis process, e.g., in synthesis by sequencing (SBS) method for sequencing of genomic sequences (Oliver Harismendy and Kelly A. Frazer, Method for improving sequence coverage uniformity of targeted genomic intervals amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology, Biotechnology, Vol. 46, No3). These processes typically may take half a day to one day to complete, and are not compatible with the point-of-care diagnostics needs Federica Pezzutol , Antonio Scarano , Carlotta Marini, Giacomo Rossi, Roberta Stocchi, Alfredo Di Cerbo and Alessandro Di Cerbo, Assessing the Reliability of Commercially Available Point of Care in Various Clinical Fields, The Open Public Health Journal, 2019, Volume 12, 342, prominent in today's rapid diagnostics culture where portable, small, low cost devices offering multiplexed analysis and short time of analysis, e.g., within 15 minutes are needed, to be performed using easily automated platforms and to be used in decentralized settings, such as urgent care clinics and pharmacies.

Accessibility of the analyte molecules toward the detector surface varies between the analytical methods, having typically a 1 D (one dimensional) access, e.g., if the surface of the detector is a surface of a vial, microtiter plate, or any passive microarray, where the binding between the molecules in the assay is controlled by diffusion of the analyte or reagent molecules and reactions toward the surface of the detector. Beads, typically 1-5 microns in diameter, when used in solutions, offer a 3 D (three dimensional) accessibility and encounters between the molecules in the solution and colloidal beads that are floating in the solution. Further accelerating a movement of magnetic detection beads by implementing magnetization on the microfluidic disposable cartridge may significantly increase the processes of the detection on the bead surfaces and potentially the sensitivity of the method. Today's trend in molecular diagnostics to provide faster responses and better direct a therapy, is to detect multiple analytes in a single test, for instance analyzing respiratory viral or bacterial panels that may consist of 10-20 pathogens to be analyzed simultaneously. The trend is even more pronounced and actual in the need for rapid, multiplexed and accurate detection of pathogens in clinical samples during pandemic outbreaks.

Multiplexed analysis of number of analytes, e.g., pathogens to recognize an infectious disease, based on magnetic beads presents a serious challenge for miniaturizing microfluidics operations and manipulations of beads in a bead array format and embedding it within the disposable microfluidic cartridge. For instance, if 20 or more analytes are to be analyzed, each bead type will carry one type of capture probes for a particular pathogen, those may require a need to add multiple reagents during sample preparation, or detection reactions. Performing multiplexed amplification reactions within the same solution has proven to provide challenges due to large number of primers needed (e.g., 20 or more pairs) that may result in highly non-specific signals. The fluidics manipulation zone to operate a single type of a bead specific for one pathogen thus may require 10-20 mm of fluid channel or space on the cartridge, which may be prohibitive in terms of its size to design a bead microarray with more than 10 detection chambers. Reducing its size, or a channel preferably to 2-5 mm would provide 5-10 fold increase in multiplexed analysis (offering approximately 50-100 analytes to be tested on the same bead array and on the same detector real estate surface. Therefore, the challenge for bead arrays to perform analysis of multiplexed analytes and at an accuracy approaching the assay statistics of a reference laboratory, is to manipulate beads accurately within very small area on the bead array detector while making contact with at least 4-6 detection solutions, including for example, ligation, amplification, reporter and washing buffer solutions. If sample preparation, or extraction of analyte from a clinical sample is embedded in the design, up to 15-18 reagents may need to be added, to perform reactions and assays at the same accuracy, LOD, sensitivity and specificity as found in the reference laboratory.

SUMMARY OF THE INVENTION

To address the foregoing problems, in whole or in part, and/or other problems that may have been observed by persons skilled in the art, the present disclosure provides compositions and methods as described by way of example as set forth below.

A microfluidic system is provided for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising:

-   -   a) a microfluidic device comprising a housing, wherein the         housing comprises a top end and a bottom end;     -   b) a plurality of reagent chambers and a plurality of         pressure-generating chambers, wherein the reagent chambers and         the pressure-generating chambers are positioned in the housing,         and wherein:         -   i) the pressure-generating chambers produce a             pressure-generating fluid using no mechanical moving parts;         -   ii) the reagent chambers are connected by at least one gas             channel at the top end of the housing to at least one of the             pressure-generating chambers;         -   iii) the reagent chambers are connected by one or more             liquid channels at the bottom end of the housing to one or             more of the pressure-generating chambers; and         -   iv) the reagent chambers comprise one or more reagent fluids             comprising one or more sets of magnetic beads comprising             capture probes specific to the one or more target analytes;     -   c) a top substrate enclosing the pressure-generating fluid         chambers, wherein the top substrate comprises fluidic channels         connecting the pressure-generating chambers to one or more vent         holes, thereby enabling movement of the one or more reagent         fluids in the one or more liquid channels at the bottom end of         the housing; and     -   d) a bottom substrate enclosing the reagent chambers; wherein         the microfluidic system is configured to achieve passive flow         resistance during filling of the microfluidic device with the         pressure-generating fluid to prevent mixing of the         pressure-generating fluid with the reagents when the         microfluidic system is not in operation; wherein the         microfluidic system is configured such that the movement of the         one or more reagent fluids is enabled by activating the one or         more pressure-generating chambers to pump the         pressure-generating fluid toward the one or more reagent         chambers and controlling and balancing pressure of the         pressure-generating fluid to achieve active flow resistance         resulting in the movement of the one or more reagent fluids in a         desired direction, whereby fluidic movement of the one or more         sets of magnetic beads is enabled and controlled; and wherein         the microfluidic system is configured to enable combining the         one or more sets of magnetic beads with one or more sample         solutions, analyte detection solutions, and/or wash solutions in         a localized area of one of the microfluidic channels, whereby         the microfluidic system is configured to magnetically release,         disperse, focus, and recapture the one or more sets of magnetic         beads, thereby enhancing magnetic bead-based detection of the         one or more target analytes. In some embodiments, achieving         passive flow resistance during filling of the microfluidic         device comprises the steps of:     -   aa) filling the plurality of pressure-generating chambers with         pressure-generating fluid;     -   bb) enclosing the housing and the plurality of         pressure-generating fluid chambers, the gas channels, and the         plurality of reagent chambers with the top substrate such that         the fluidic channels make desired connections between the         chambers and vent holes enabling movement of one or more reagent         fluids in liquid channels at the bottom end of the housing; and     -   cc) inverting the microfluidic device and filling the plurality         of reagent chambers with the one or more reagent fluids and         enclosing the reagent chambers, the one or more reagent fluids,         and the liquid channels with a bottom substrate at the bottom         end of the housing.

In some embodiments, the microfluidic system further comprises an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.

In some embodiments, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

In some embodiments, the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.

In some embodiments, the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.

In some embodiments, the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.

In some embodiments, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.

In some embodiments, the microfluidic system further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments, the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.

In some embodiments, the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.

In some embodiments, the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.

In some embodiments, the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes.

In some embodiments, the microfluidic system further comprises vibrating micromotors configured to enhance convective transport and mixing.

A method is also provided for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising:

-   -   a) providing a microfluidic system comprising a microfluidic         device, wherein the microfluidic device comprises:         -   i) a housing, wherein the housing comprises a top end and a             bottom end;         -   ii) a plurality of reagent chambers and a plurality of             pressure-generating chambers, wherein the reagent chambers             and the pressure-generating chambers are positioned in the             housing, and wherein:             -   aa) the pressure-generating chambers produce a                 pressure-generating fluid using no mechanical moving                 parts;             -   bb) the reagent chambers are connected by at least one                 gas channel at the top end of the housing to at least                 one of the pressure-generating chambers;             -   cc) the reagent chambers are connected by one or more                 liquid channels at the bottom end of the housing to one                 or more of the pressure-generating chambers; and             -   dd) the reagent chambers comprise one or more reagent                 fluids comprising one or more sets of magnetic beads                 comprising capture probes specific to the one or more                 target analytes;         -   iii) a top substrate enclosing the pressure-generating fluid             chambers, wherein the top substrate comprises fluidic             channels connecting the pressure-generating chambers to one             or more vent holes, thereby enabling movement of one or more             reagent fluids in the one or more liquid channels at the             bottom end of the housing; and             -   iv) a bottom substrate enclosing the reagent chambers;             -   wherein the microfluidic system is configured to achieve                 passive flow resistance during filling of the                 microfluidic device with the pressure-generating fluid                 to prevent mixing of the pressure-generating fluid with                 the reagents when the microfluidic system is not in                 operation;     -   b) activating the one or more pressure-generating chambers to         pump the pressure-generating fluid toward the one or more         reagent chambers and controlling and balancing pressure of the         pressure-generating fluid to achieve active flow resistance         resulting in the movement of the one or more reagent fluids in a         desired direction, whereby fluidic movement of the one or more         sets of magnetic beads is enabled and controlled; and     -   c) combining the one or more sets of magnetic beads with one or         more sample solutions, analyte detection solutions, and/or wash         solutions in a localized area of one of the microfluidic         channels, and magnetically releasing, dispersing, focusing, and         recapturing the one or more sets of magnetic beads, thereby         enhancing magnetic bead-based detection of the one or more         target analytes.

In some embodiments, the method for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels further comprises achieving passive flow resistance during filling of the microfluidic device, further comprising the steps of:

-   -   ai) filling the plurality of pressure-generating chambers with         pressure-generating fluid;     -   bi) enclosing the housing and the plurality of         pressure-generating fluid chambers, the gas channels, and the         plurality of reagent chambers with the top substrate such that         the fluidic channels make desired connections between the         chambers and vent holes enabling movement of one or more reagent         fluids in liquid channels at the bottom end of the housing; and     -   ci) inverting the microfluidic device and filling the plurality         of reagent chambers with the one or more reagent fluids and         enclosing the reagent chambers, the one or more reagent fluids,         and the liquid channels with a bottom substrate at the bottom         end of the housing.

In some embodiments, the method is executed by an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.

In some embodiments of the method, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

In some embodiments of the method, the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.

In some embodiments of the method, the one or more reagent fluids comprise one or more reagents for extraction, amplification, or detection, comprising one or more biomarkers, nutrients, and/or chemicals.

In some embodiments of the method, the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.

In some embodiments of the method, the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.

In some embodiments of the method, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.

In some embodiments of the method, the microfluidic device further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments of the method, the microfluidic system is configured to enable the gas produced by electrolysis to control pH and/or conductivity reactions in the one or more of the plurality of reagent chambers.

In some embodiments of the method, the microfluidic system further comprises one or more gas permeable membranes atop the plurality of pressure generation chambers, wherein the one or more gas permeable membranes separate liquid and gas pressure-generating fluids in the pressure-generating chambers while allowing permeation of pressure-generating fluid into the fluidic channels without mixing between the pressure-generating fluid and the one or more reagent fluids in the plurality of reagent chambers.

In some embodiments of the method, the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.

In some embodiments of the method, the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.

In some embodiments of the method, the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.

In some embodiments of the method, the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes.

In some embodiments of the method, the microfluidic system further comprises vibrating micromotors configured to enhance convective transport and mixing.

Additional features of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims.

To address the foregoing problems, in whole or in part, and/or other problems that may have been observed by persons skilled in the art, the present disclosure provides compositions and methods as described by way of example as set forth below.

A microfluidic system is provided for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising:

-   -   a) a microfluidic device comprising a housing, wherein the         housing comprises a top end and a bottom end;     -   b) a plurality of reagent chambers and a plurality of         pressure-generating chambers, wherein the reagent chambers and         the pressure-generating chambers are positioned in the housing,         and wherein:         -   i) the pressure-generating chambers produce a             pressure-generating fluid using no mechanical moving parts;         -   ii) the reagent chambers are connected by at least one gas             channel at the top end of the housing to at least one of the             pressure-generating chambers;         -   iii) the reagent chambers are connected by one or more             liquid channels at the bottom end of the housing to one or             more of the pressure-generating chambers; and         -   iv) the reagent chambers comprise one or more reagent fluids             comprising one or more sets of magnetic beads comprising             capture probes specific to the one or more target analytes;     -   c) a top substrate enclosing the pressure-generating fluid         chambers, wherein the top substrate comprises fluidic channels         connecting the pressure-generating chambers to one or more vent         holes, thereby enabling movement of the one or more reagent         fluids in the one or more liquid channels at the bottom end of         the housing; and     -   d) a bottom substrate enclosing the reagent chambers;

wherein the microfluidic system is configured to achieve passive flow resistance during filling of the microfluidic device with the pressure-generating fluid to prevent mixing of the pressure-generating fluid with the reagents when the microfluidic system is not in operation; wherein the microfluidic system is configured such that the movement of the one or more reagent fluids is enabled by activating the one or more pressure-generating chambers to pump the pressure-generating fluid toward the one or more reagent chambers and controlling and balancing pressure of the pressure-generating fluid to achieve active flow resistance resulting in the movement of the one or more reagent fluids in a desired direction, whereby fluidic movement of the one or more sets of magnetic beads is enabled and controlled; and wherein the microfluidic system is configured to enable combining the one or more sets of magnetic beads with one or more sample solutions, analyte detection solutions, and/or wash solutions in a localized area of one of the microfluidic channels, whereby the microfluidic system is configured to magnetically release, disperse, focus, and recapture the one or more sets of magnetic beads, thereby enhancing magnetic bead-based detection of the one or more target analytes. In some embodiments, achieving passive flow resistance during filling of the microfluidic device comprises the steps of:

-   -   aa) filling the plurality of pressure-generating chambers with         pressure-generating fluid;     -   bb) enclosing the housing and the plurality of         pressure-generating fluid chambers, the gas channels, and the         plurality of reagent chambers with the top substrate such that         the fluidic channels make desired connections between the         chambers and vent holes enabling movement of one or more reagent         fluids in liquid channels at the bottom end of the housing; and     -   cc) inverting the microfluidic device and filling the plurality         of reagent chambers with the one or more reagent fluids and         enclosing the reagent chambers, the one or more reagent fluids,         and the liquid channels with a bottom substrate at the bottom         end of the housing.

In some embodiments, the microfluidic system further comprises an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.

In some embodiments, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

In some embodiments, the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.

In some embodiments, the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.

In some embodiments, the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.

In some embodiments, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.

In some embodiments, the microfluidic system further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments, the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.

In some embodiments, the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.

In some embodiments, the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.

In some embodiments, the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes.

In some embodiments, the microfluidic system further comprises vibrating micromotors configured to enhance convective transport and mixing.

A method is also provided for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising:

-   -   a) providing a microfluidic system comprising a microfluidic         device, wherein the microfluidic device comprises:         -   i) a housing, wherein the housing comprises a top end and a             bottom end;         -   ii) a plurality of reagent chambers and a plurality of             pressure-generating chambers, wherein the reagent chambers             and the pressure-generating chambers are positioned in the             housing, and wherein:             -   aa) the pressure-generating chambers produce a                 pressure-generating fluid using no mechanical moving                 parts;             -   bb) the reagent chambers are connected by at least one                 gas channel at the top end of the housing to at least                 one of the pressure-generating chambers;             -   cc) the reagent chambers are connected by one or more                 liquid channels at the bottom end of the housing to one                 or more of the pressure-generating chambers; and             -   dd) the reagent chambers comprise one or more reagent                 fluids comprising one or more sets of magnetic beads                 comprising capture probes specific to the one or more                 target analytes;         -   iii) a top substrate enclosing the pressure-generating fluid             chambers, wherein the top substrate comprises fluidic             channels connecting the pressure-generating chambers to one             or more vent holes, thereby enabling movement of one or more             reagent fluids in the one or more liquid channels at the             bottom end of the housing; and         -   iv) a bottom substrate enclosing the reagent chambers;         -   wherein the microfluidic system is configured to achieve             passive flow resistance during filling of the microfluidic             device with the pressure-generating fluid to prevent mixing             of the pressure-generating fluid with the reagents when the             microfluidic system is not in operation;     -   b) activating the one or more pressure-generating chambers to         pump the pressure-generating fluid toward the one or more         reagent chambers and controlling and balancing pressure of the         pressure-generating fluid to achieve active flow resistance         resulting in the movement of the one or more reagent fluids in a         desired direction, whereby fluidic movement of the one or more         sets of magnetic beads is enabled and controlled; and     -   c) combining the one or more sets of magnetic beads with one or         more sample solutions, analyte detection solutions, and/or wash         solutions in a localized area of one of the microfluidic         channels, and magnetically releasing, dispersing, focusing, and         recapturing the one or more sets of magnetic beads, thereby         enhancing magnetic bead-based detection of the one or more         target analytes.

In some embodiments, the method for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels further comprises achieving passive flow resistance during filling of the microfluidic device, further comprising the steps of:

-   -   ai) filling the plurality of pressure-generating chambers with         pressure-generating fluid;     -   bi) enclosing the housing and the plurality of         pressure-generating fluid chambers, the gas channels, and the         plurality of reagent chambers with the top substrate such that         the fluidic channels make desired connections between the         chambers and vent holes enabling movement of one or more reagent         fluids in liquid channels at the bottom end of the housing; and     -   ci) inverting the microfluidic device and filling the plurality         of reagent chambers with the one or more reagent fluids and         enclosing the reagent chambers, the one or more reagent fluids,         and the liquid channels with a bottom substrate at the bottom         end of the housing.

In some embodiments, the method is executed by an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.

In some embodiments of the method, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

In some embodiments of the method, the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.

In some embodiments of the method, the one or more reagent fluids comprise one or more reagents for extraction, amplification, or detection, comprising one or more biomarkers, nutrients, and/or chemicals.

In some embodiments of the method, the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.

In some embodiments of the method, the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.

In some embodiments of the method, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.

In some embodiments of the method, the microfluidic device further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments of the method, the microfluidic system is configured to enable the gas produced by electrolysis to control pH and/or conductivity reactions in the one or more of the plurality of reagent chambers.

In some embodiments of the method, the microfluidic system further comprises one or more gas permeable membranes atop the plurality of pressure generation chambers, wherein the one or more gas permeable membranes separate liquid and gas pressure-generating fluids in the pressure-generating chambers while allowing permeation of pressure-generating fluid into the fluidic channels without mixing between the pressure-generating fluid and the one or more reagent fluids in the plurality of reagent chambers.

In some embodiments of the method, the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.

In some embodiments of the method, the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.

In some embodiments of the method, the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.

In some embodiments of the method, the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes.

In some embodiments of the method, the microfluidic system further comprises vibrating micromotors configured to enhance convective transport and mixing.

Additional features of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the subject matter of the present invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:

FIGS. 1A-1D are simplified cross-sectional view illustrations of the microfluidic device constructed and operative showing fluidic channel for performing magnetic separations of magnetic beads using conventional methods.

FIGS. 1E and 1F are simplified cross-sectional view illustrations of the microfluidic device of the invention for implementation of combined magnetic field, electric field enhancement of processes in bead array microfluidic system.

FIGS. 1G-1J are simplified cross-sectional view illustrations of the microfluidic device of the invention for implementation of combined magnetic field and electric field wherein magnetic and electric field lines are under different angles and in correlation to fluid flow direction promoting enhancement of processes in bead array channels of the microfluidic system of the invention.

FIG. 1K shows enhancement of encounters of molecules and detection magnetic beads in an embodiment wherein the magnetic beads are accumulated by a magnet positioned on top of the fluidic channel and focused by magnetic field lines onto a window, wherein the electric field lines are at a right angle to the magnetic field lines and opposite to the fluid flow direction.

FIG. 2A is a top view of the housing and microfluidic channels and chambers of the microfluidic device showing components of the electrolytic fluid pumping and bead array chamber.

FIGS. 2B-2F are 3D (three dimensional) illustrations of the structure of manufactured microfluidic device of the invention for implementation of combined magnetic field, electric field and convective enhancement of processes in bead array microfluidic system. FIG. 2B shows single lane bead array, FIG. 2C shows multiple (three) lane array with multiple bead array chambers, FIG. 2D. shows multiple (three) lane array with multiple bead array chambers and a focusing magnet in support of localized magnetization over the array, FIG. 2E is a closeup of electrolytic pump, reagent chamber with beads, and FIG. 2F is further close up of the bead array chamber.

FIGS. 3A-3E are schematic illustrations of the operation steps of the microfluidic device of the invention for implementation of combined magnetic field, electric field and convective enhancement of processes in preferred bead array microfluidic system.

FIGS. 4A-4D are schematic illustrations of the control of reactions in the microfluidic device of the invention for implementation of combined magnetic field, electric field, and convective processes to enhance different reactions: FIG. 4A: target capturing, and FIGS. 4B-4D show steps of rolling circle isothermal amplification (RCA) of nucleic acid targets on bead array.

FIGS. 5A-5C show experimental data for performing RCA amplification of Hemophilus influenzae DNA target on bead array; FIG. 5A—photographs of fluorescence detection in presence and absence of target; FIG. 5B—plot of fluorescence profile, and FIG. 5C graphical presentation and quantitation of data.

FIGS. 6A-6C shows repeat experimental data for performing RCA amplification of Hemophilus influenzae DNA target on bead array and performing fluorescence analysis on glass slides; FIG. 6A—photographs of fluorescence detection in presence and absence of target; FIG. 6B—plot of fluorescence profile, and FIG. 6C graphical presentation and quantitation of data.

FIGS. 7A-7C shows experimental data for performing RCA amplification using real-time reporting fluorescence-quencher probe of Acinetobacter baumanii DNA target on bead array; FIG. 7A—photographs of fluorescence detection in presence and absence of target; FIG. 7B—plot of fluorescence profile, and FIG. 7C graphical presentation and quantitation of data.

FIGS. 8A-8C show experimental data demonstrating enhancement of combined electric+magnetic field vs. magnetic field only applied to target capture using DNA target and performing a comparison between the reactions: FIG. 8A—photographs of fluorescence detection in presence and absence of target; FIG. 8B—plot of fluorescence profile, and FIG. 8C graphical presentation and quantitation of data.

FIGS. 9A-9D are photographs of microfluidic steps operating the microfluidic device of the invention for implementation of combined magnetic field, electric field and convective enhancement of processes in bead array microfluidic system using fluorescently labeled beads to confirm operation of each step in the device.

FIG. 10 shows assay protocols and data for detection and identification of Escherichia coli pathogen in whole blood clinical samples on a microfluidic device according to an embodiment of the invention. FIGS. 10A-10C show a combination of system operations shown in FIG. 11, where the magnetic beads are disposed onto electrodes and kept focused under magnetic field. The electric field is implemented as shown in FIG. 1K, where stainless steel electrodes, circular working electrodes with beads, and counter electrodes of same stainless steel material are configured as a planar array.

FIG. 11 is a block diagram illustrating an example wired or wireless processor enabled device that may be used in connection with various embodiments described herein.

DETAILED DESCRIPTION

The subject matter of the present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the subject matter of the present invention are shown. Like numbers refer to like elements throughout. The subject matter of the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the subject matter of the present invention set forth herein will come to mind to one skilled in the art to which the subject matter of the present invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. All illustrations of the drawings are for the purpose of describing selected versions of the present invention and are not intended to limit the scope of the present invention. Therefore, it is to be understood that the subject matter of the present invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

System for Combined Electric, Magnetic, and Convective Acceleration of Chemical and Biochemical Reactions and Methods of use Thereof

The present invention utilizes a multiple electrolytic pumps-based system to enhance reactions on magnetic beads by combining magnetic field, electric field and convective acceleration of processes and reactions in bead-array chambers. The electrolytic generation of gases, like oxygen and hydrogen generated from electrolysis of aqueous, preferably salt solutions, is used as a pumping fluid in pressure generating chambers to pressurize the fluid in reagent chambers and fluidic channels and move the fluid of interest, from one reagent chamber to another, in a desired direction. The desired direction may include back-and-forth movement of one or more fluids of interest in the microfluidic device, enabling mixing between reagents in different reagent chambers.

Accordingly, in one embodiment, a microfluidic system is provided for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising:

-   -   a) a microfluidic device comprising a housing, wherein the         housing comprises a top end and a bottom end;     -   b) a plurality of reagent chambers and a plurality of         pressure-generating chambers, wherein the reagent chambers and         the pressure-generating chambers are positioned in the housing,         and wherein:         -   i) the pressure-generating chambers produce a             pressure-generating fluid using no mechanical moving parts;         -   ii) the reagent chambers are connected by at least one gas             channel at the top end of the housing to at least one of the             pressure-generating chambers;         -   iii) the reagent chambers are connected by one or more             liquid channels at the bottom end of the housing to one or             more of the pressure-generating chambers; and         -   iv) the reagent chambers comprise one or more reagent fluids             comprising one or more sets of magnetic beads comprising             capture probes specific to the one or more target analytes;     -   c) a top substrate enclosing the pressure-generating fluid         chambers, wherein the top substrate comprises fluidic channels         connecting the pressure-generating chambers to one or more vent         holes, thereby enabling movement of the one or more reagent         fluids in the one or more liquid channels at the bottom end of         the housing; and     -   d) a bottom substrate enclosing the reagent chambers;         wherein the microfluidic system is configured to achieve passive         flow resistance during filling of the microfluidic device with         the pressure-generating fluid to prevent mixing of the         pressure-generating fluid with the reagents when the         microfluidic system is not in operation; wherein the         microfluidic system is configured such that the movement of the         one or more reagent fluids is enabled by activating the one or         more pressure-generating chambers to pump the         pressure-generating fluid toward the one or more reagent         chambers and controlling and balancing pressure of the         pressure-generating fluid to achieve active flow resistance         resulting in the movement of the one or more reagent fluids in a         desired direction, whereby fluidic movement of the one or more         sets of magnetic beads is enabled and controlled; and wherein         the microfluidic system is configured to enable combining the         one or more sets of magnetic beads with one or more sample         solutions, analyte detection solutions, and/or wash solutions in         a localized area of one of the microfluidic channels, whereby         the microfluidic system is configured to magnetically release,         disperse, focus, and recapture the one or more sets of magnetic         beads, thereby enhancing magnetic bead-based detection of the         one or more target analytes. In some embodiments, achieving         passive flow resistance during filling of the microfluidic         device comprises the steps of:     -   aa) filling the plurality of pressure-generating chambers with         pressure-generating fluid;     -   bb) enclosing the housing and the plurality of         pressure-generating fluid chambers, the gas channels, and the         plurality of reagent chambers with the top substrate such that         the fluidic channels make desired connections between the         chambers and vent holes enabling movement of one or more reagent         fluids in liquid channels at the bottom end of the housing; and     -   cc) inverting the microfluidic device and filling the plurality         of reagent chambers with the one or more reagent fluids and         enclosing the reagent chambers, the one or more reagent fluids,         and the liquid channels with a bottom substrate at the bottom         end of the housing.

Each reagent chamber may be connected to at least one or more pressure generating chambers enabling balancing of pressures in fluidic channels, thus actively controlling the resistance to flow in fluidic channels, and by controlling intensity and timing of the pressure generation in operating pressure generating chambers. This results in directing the fluid flow through desired channels or reagent chambers and in a desired direction. Actuating particular pressure generation actuators, by, e.g., starting the electrolysis in one or more pressure generating chambers within pressure generating fluid and producing and moving a pumping fluid (gas, liquid, oil) at controlled voltage or current applied through the electrodes embedded in the pressure generation chamber, can define the fluidic protocol to operate multiple fluidic steps in the microfluidic device.

Additionally, or alternatively, one or more fluids can be moved simultaneously, in parallel, or in a series of fluidic steps within the housing of the microfluidic device. This is achieved by activating pressure generation actuators, e.g., initiating electrolysis in one or more pressure generation chambers and controlling the intensity of gas evolution and timing of evolution. According to basic Faraday and Nernst equations of electrochemical splitting of water (or other pressure generating fluid), the current applied on electrodes is proportional to the number of moles of gas produced which is further proportional to the pressure of gas produced. Very small amount of water can produce large volumes of pressurized electrolytic gas, e.g., 1 mol of water, or 18 g or 18 mL of water produce 22.4 liters of gas (in accordance with Ideal Gas Law). This enables that just a few hundred microliters of pressure generating fluid stored in pressure generating chambers will produce large amounts and enough volume of pressurized gas to run the microfluidic device operations for long time. Pressures of up to several hundred psi can be produced electrolytically, depending on the fluidics design, the chamber and channels geometry in the device, and current intensity applied within an operation time of the microfluidic device.

The same electrolytic actuation of pressure generation provides an option to produce minute quantities, and pressures of gas, thus enabling a very slow and highly controlled movement of fluids in the channels or reagent chambers. Such slow, precise flows are useful in controlling slower reactions in chemical and biochemical applications of the microfluidic device and bead array processes, for instance, but not limited to sample preparation or analyte detection using controlled movement between analyte target and detector, or in dispersion and concentration of beads, including magnetic beads in the fluidic channels. In some embodiments, the microfluidic system further comprises an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device. The computerized control of the actuators producing pressure in different regions of the microfluidic device and establishing protocols for applying varying pressures in multiple pressure reagent chambers and balancing pressures so that the resistance to flow in fluidic channels is actively controlled may be essential in reproducibly operating fluidic protocols in disposable cartridges.

In some embodiments, the pressure-generating fluid may comprise at least one or more liquid, oil, gas, or air fluids. The pressure-generating fluid, for instance salty water in the electrolytic actuation of pressure, can be oil, lighter or heavier than water that is pushed into other channels of the microfluidic device of the invention to modify the resistance to flow in the channel and act as a valving mechanism where fluid flow in such higher resistance oil filled channel will be prevented, and allowed in a channel of lower resistance. The active control of resistance includes increasing a resistance to flow in a particular channel where gas or air pressure-generating fluids are pumped into liquid fluidic channels generating bubbles between the reagent chambers and actively affecting the flow resistance in said channel, further providing means of valving or flow control. Especially in a Y shaped design of fluidic channels, where a decision is needed in which direction the fluid should flow, exiting through the Y channel split, the injection of a different fluid phase, such as a gas into liquid, or oil into liquid, an accurate control or resistance is achieved in this manner and can be used to direct a first fluid from one reagent chamber exiting left in the Y design, and a second fluid from a different reagent chamber exiting right into a different section of the fluidic chambers in the microfluidic device of the invention. Such splitting of channels is useful for instance when sample preparation or detection processes are performed in the analytical, diagnostics applications of the microfluidic device of the invention, where washing solutions are sent into a waste chamber, and eluent or detection/analyte solution over a detection chamber or sensor.

In some embodiments, the microfluidic system further comprises an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.

In some embodiments, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

In some embodiments, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution. In some embodiments, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

Accordingly, there is further provided in accordance with yet another embodiment of the present invention a microfluidic device wherein actuation of a pressure in said pressure-generating chambers is generated using thermal heating, utilizing electrodes or coils position directly within the pressure-generating fluid, or heaters including, but not limited to screen-printed inks at the bottom substrate of the housing, in locations where heating or pressure generation is desired, such as in pressure-generating chambers. Typically, highly conductive screen-printed, meandering coils, based on conductive silver inks can be printed on pressure sensitive adhesive and bonded to the bottom substrates of the device housing. The contacts to these heating elements may be provided directly by contacting the silver ink printed lines at the edge of the device housing using spring loaded pogo-pins, or inserting the housing with printed silver ink contacts into a terminal located within the instrument or microfluidics controller. Alternatively, a simple, low cost, printed circuit board (PCB) with copper lines can be attached to the bottom of the housing of the device where either pin contacts or screen-printed conductive lines are pressed with the PCB board. The contact to the PCB board and contact lines are made using standard electronic terminals located in the instrument or microfluidics controller.

There is also provided in accordance with another embodiment of the present invention a microfluidic system based on active control of flow resistance in microfluidic channels wherein actuation of a pressure in said pressure-generating chambers is generated using catalytic heating, utilizing hydrogen gas produced electrolytically in the pressure producing chamber and passing the hydrogen over a miniature catalytic converter, where catalyst chosen from, but not limited to Pt, Pd, particles or deposits is made on a ceramic substrate. The size of such catalytic converter is preferably 1-20 mm. the hydrogen gas passing over the catalyst heats up the ceramic insert in the housing of the microfluidic device and rapidly generating the heat, and subsequently the vapors created generate pressure in the fluidic channel. Such catalytic heating using hydrogen passing over the miniature catalytic converter can heat the miniature ceramic element to 600 C within only 3-5 seconds. The temperature is controlled by the amount of hydrogen produced electrolytically, which is further controlled electronically by adjusting the current or voltage on the electrolytic electrodes in the pressure-generating chamber.

In accordance with yet another embodiment of the present invention, a microfluidic system based on active control of flow resistance in microfluidic channels wherein actuation of a pressure in said pressure-generating chambers is generated using ultrasonically created pressure. The pressure is generated using ultrasonic piezoelectric transducers that under an applied high-frequency alternating voltage pulses contract or expand generating mechanical vibrations that serve as pressure generation for movement of fluids in the fluidic channels or reagent chambers of the present invention. Typically artificially manufactured piezoelectric materials such as, but not limited to Polyvinylidene difluoride, PVDF or PVF2, Barium titanate, Lead titanate, Lead zirconate titanate (PZT), Potassium niobate, Lithium niobate or Lithium tantalate are used as piezoelectric elements activated by electrodes in contact with the piezoelectric material.

In some embodiments, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine. For example, an electrolytic generation of gases, like oxygen and hydrogen generated from electrolysis of aqueous, particularly salt solutions, may be used as a pressure-generating fluid in pressure-generating chambers to pressurize the fluid in reagent chambers and fluidic channels and move the fluid of interest, from one reagent chamber to another, in a desired direction. The desired direction may include back-and-forth movement of one or more fluids of interest in the microfluidic device, enabling mixing between reagents in different reagent chambers.

Accordingly, in another embodiment, pressure-generating fluids other than water, but not limited to salt solutions are used, e.g., containing chlorides, carbonates or other salts that will produce gases in addition or other than oxygen and hydrogen from water splitting. Thus, chloride solutions will produce chlorine gas, carbonate solution carbon dioxide at lower pH, and other reactions known in the art that could be utilized to generate gases useful not only in controlling pressures in the fluidic channels but actively controlling reactions in channels or chambers. Such embodiments of the present invention that include active, or on demand production of reactant gases, or reactants for controlling reactions in reagent chambers, may include, but are not limited to, controlling pH in reagent chambers, through using anolyte and catholyte solution from pressure pumping chambers and fluids, that generate acidic (where oxygen is evolved) or basic (where hydrogen is evolved) solutions or reactant that can adjust a pH in the reagent chamber, or chlorine for disinfection of the device, e.g., post-using steps that involve infectious agents in the device, or oxygen to control aerobic growth of cells, pathogens, or organoids, or carbon dioxide to control anaerobic growth of cells, pathogens, or organoids in the various applications of the fluidic device and system of present invention.

In some embodiments, the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.

In some embodiments, the microfluidic system further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments, the microfluidic system is configured to enable the gas produced by electrolysis to control pH and/or conductivity reactions in the one or more of the plurality of reagent chambers.

In some embodiments, the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.

In some embodiments, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.

In some embodiments, the microfluidic system further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments, the one or more reagent fluids comprise aqueous or non-aqueous liquids comprising one or more reagents for extraction, amplification, or detection of one or more analytes comprising one or more biomarkers, nutrients, and/or chemicals. The reagent fluids may comprise any sample that comprises one or more biomarkers, nutrients, and/or chemicals, such as an analytic sample, clinical sample, and the like. The one or more biomarkers may comprise any nucleic acid (DNA or RNA), protein, or fragments thereof. The one or more chemicals may comprise: chemicals for analyte extraction, amplification, and/or detection, chemicals useful in controlling fluids in microfluidic devices or cartridges; nutrients for controlling growth of cells, pathogens, and/or organoids (e.g., including but not limited to tissue engineering or cloning processes); chemicals as reagents for generating inorganic and organic compounds (e.g., including but not limited to inorganic crystals or protein crystallization); and/or chemicals for generating nano-compounds or nano-elements (e.g., including but not limited to carbon nanotubes, nanofilaments, and/or graphene compounds).

In some embodiments, the microfluidic system further comprises one or more gas permeable membranes atop the plurality of pressure generation chambers, wherein the one or more gas permeable membranes separate liquid and gas pressure-generating fluids in the pressure-generating chambers while allowing permeation of pressure-generating fluid into the fluidic channels without mixing between the pressure-generating fluid and the one or more reagent fluids in the plurality of reagent chambers.

In some embodiments, the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.

In some embodiments, the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.

In some embodiments, the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions. The magnetic field and electric field that enhance contact of the magnetic beads with the reagents or the analyte molecules brought into said magnetic beads detection channels by said fluid flow can be applied under an angle, where the angle between the magnetic field and electric field can be 180°, causing the magnetic and electric field lines being in the opposing directions, 90° causing said magnetic and electric field line to be under right angle, and in parallel or opposing said fluid flow direction. Other angles between said magnetic and electric field may be also used, as will be clear to those skilled in the art by which contacting and interactions between said magnetic detection beads driven by said magnetic field and said reagent or analyte molecules driven by said electric field can be enhanced within small volumes within said magnetic beads channels.

In some embodiments, the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes. Embodiments of present invention enable efficient mixing and for reactions to occur on the surface of said magnetic beads within channel volumes as small as 0.1 microliter (when thickness of the channel is in the range of 0.1 mm and the zone of said magnetic bead collection are about 1 mm×1 mm). Multiplexed detection is enabled on said sets of magnetic beads each having different probes specific for a different analyte. Thus, said analyte sample brought into said magnetic beads channel by said fluid flow is interrogated simultaneously and spatially (at distances as small but not limited to 0.5-1 mm) by each set of magnetic beads and within small, microliter volumes of said sample where said magnetic beads are manipulated magnetically and reagent and analyte molecules are manipulated electrically.

In another embodiment, said electric field can be a planar field, wherein said electrodes producing said electric field are positioned in the same plane, for instance at the bottom of said magnetic bead detection channels, and said electric field lines protrude vertically into said channel and through said fluid flow. This embodiment enables magnetic beads to be focused or transported by magnetic field at the bottom of said magnetic bead channel or at the top of the channel. In the case that said magnetic field focuses or accumulates said magnetic beads at the bottom of said channel, and on said electrodes producing said electric field, the analyte and/or reagent molecules are directed by the electric field onto the surface of planar electrodes and enable enhanced accumulation and contact on the surface of said magnetic beads. Typically, a detection of the analyte accumulated on the surface of said magnetic beads by said electric field could occur after introduction of a reporter reagent by said fluid flow reporting only analyte capturing on specific probes on said magnetic beads, specific to a particular analyte. Multiplexed detection can be obtained by using multiple said electrodes producing said electric field in said magnetic bead channels where each set of said beads is accumulated on one set of electrodes and having probes specific for a different analyte. Typically, an optical detection, such as, but not limited to fluorescence detection is used to simultaneously detect all multiplexed analytes on multiple sets of said beads.

In yet another embodiment, wherein said electric field is a planar field, and said electrodes producing said electric field are positioned in the same plane, and wherein said magnetic beads are focused or accumulated at the top surface of said bead detection channels by bringing an external magnet at the top surface of said bead detection channel and said electric field lines protrude vertically into said channel and through said fluid flow bringing electrically vertically said analyte molecules to the top of said channel and onto surfaces of said magnetic detection beads. The magnets in such embodiment can be removed before optical detection, and images taken through a transparent window covering the top of said magnetic beads detection channel.

There is also provided in accordance with another preferred embodiment of the present invention a microfluidic system wherein said magnetic beads are deposited or spotted onto said electrodes producing said planar electric field in said magnetic beads detection channel. Said sets of magnetic beads, each containing one type of capture probes specific for one said analyte, are spotted, manually or using robotized spotting onto different spots of said magnetic beads or said electrodes. Focusing of said sets of magnetic beads during the manufacturing of said magnetic beads can be aided with a magnetic array having miniature magnets with a diameter corresponding to a diameter of said electric field electrodes. Such magnetic beads and a cartridge manufacturing process can include laminating a low-cost disposable magnetic strip magnet that serves for preserving and keeping spotted magnetic beads in its location on said magnetic beads and said electrodes during storage and during operation when said fluid flow brings said analyte solution and different reagent solutions.

Magnetic bead detection of one or more reagents and/or analytes can typically contain streptavidin to bind biotinylated probes specific to each analyte on said magnetic beads. Other reagents, such as forward and reverse primer, or probes enabling amplification of specifically captured analyte can be embedded on said magnetic beads and enable various biochemical reactions that enhance detection, such as, but not limited to hybridization reactions, ligation, polymerization, specific binding and extraction or enrichment of said analyte molecules. Both nucleic acid and immuno- analyte targets can be used in the detection and simultaneously.

The present invention enables simultaneous dual, triple, or greater multiples of analysis on the same magnetic beads in a disposable cartridge. Dual molecular (for nucleic acids) and immuno-detection (for proteins, including but not limited to antibodies and antigens) can be performed simultaneously. Sensitivity of immuno-detection can be further enhanced by performing amplification reactions, for instance through binding the secondary, target recognition antibody with a nucleic acid oligonucleotide and performing isothermal amplification of bound oligonucleotides.

The present invention involves the use of various isothermal nucleic acid amplification methods performed in situ on surfaces of said detection magnetic beads and enabling highly multiplexed and sensitive detection of both nucleic acid and immuno- analyte targets. Such detection methods include analyte amplification reactions but not limited to PCR, real- time PCR, isothermal amplifications such as but not limited to LAMP, RCA, NASBA, TMA, specific binding and capturing of analytes such as, but not limited to Target Capture and capturing of molecules such as, but not limited to nucleic acids, proteins and chemical analytes.

In another embodiment of the present invention, a microfluidic system is provided wherein the magnetic beads are deposited, spotted, or sequestered into a storage chamber. Each set of magnetic beads is deposited, spotted, or sequestered into one of the miniature storage chambers, typically indentations in the cartridge surface or small holes, e.g., containing 1-5 uL of bead solution, drilled at envisioned locations in the microfluidic channel. To preserve the capture probes on the magnetic beads, the magnetic beads are dried or embedded into to raffinose or hydrogels or the like, or bonded to the surface using covalent or other bonding techniques. During activation and detection, fluids comprising one or more analytes and one or more reagents are brought over the beads by fluid flow over or in the storage chambers. The magnetic beads are mobilized by the fluid flow but are immediately captured by a magnetic field through a magnetic array positioned at the top of the microfluidic channel, the magnets of the array having a small diameter, about 0.5-3 mm, corresponding to the accumulation spots for magnetic beads. The magnetic beads are lifted vertically to the top of the magnetic bead channel. Each set of the magnetic beads is lifted vertically simultaneously, but separately from each other, with no mixing between the sets of the magnetic beads. Accumulation or focusing of the magnetic beads is made on the top window and allows for moving the accumulated beads along the window to a location where optical imaging is performed toward the end of the assay protocol.

In some embodiments, the microfluidic system further comprises vibrating micromotors configured to enhance convective transport and mixing. Reference is now made to FIGS. 1A-1D which are simplified cross-sectional view illustrations of the microfluidic device constructed and operative showing fluidic bottom 101 and top 102 of a fluidic channel 100 for performing magnetic separations of magnetic beads 103 using conventional methods. A magnet 106 is brought in close contact with the bottom of fluidic channel, typically enclosed in a microfluidic housing in direction 108. Magnetic beads 103 move within the solution toward the bottom wall 101 of the fluidic channel 100 and concentrate there. If a magnet is moved toward the top of the fluidic channel 102 it moves in the opposite direction, demonstrating conventional principles of magnetic bead accumulation.

Reference is now made to FIGS. 1E and 1F which are simplified cross-sectional view illustrations of the microfluidic device of the invention for implementation of combined magnetic field, electric field enhancement of processes in bead array microfluidic system. Compared to conventional systems shown in FIGS. 1A-1D, the fluidic channel 100 or chamber for bead array operation additionally contains electrodes 118 connecting to positive contact 122 of an external power supply and negative electrode 120 connected to negative contact 124. The electric field induced in the fluidic channel 100 causes ions, anions 126 to move toward the positive electrode 118 in direction 128, and cations 130 to move toward negative electrode 120 in direction 132. Analyte molecules, for instance, but not limited to nucleic acids, DNA and RNA, or proteins, if negatively charged will move toward positive electrode 118. FIG. 1F demonstrates the situation in which magnetic beads 116 move toward the bottom end 101 of the fluidic channel 100 when the magnet 106 is put in proximity as shown in direction 108. In this example, the magnetic field moves magnetic bead down 136, and the electric field in the opposite direction 138. Such intense contacting and increasing accessibility or magnetic beads to analyte molecules 134 significantly enhances the reactions occurring on moving magnetic beads, minimizing any slow diffusion process that typically occur in solution if no stirring or motion is implemented in the reaction chambers or fluidic channels. Fluidic movement by itself cannot support increase of the reaction rates in the channels since a Laminar flow, no stirring or turbulence, is typical in channels of microfluidics devices. Reference is now made to FIGS. 1G-1J which are simplified cross-sectional view illustrations of the microfluidic device of the invention for implementation of combined magnetic field and electric field wherein magnetic and electric field lines are under different angles and in correlation to fluid flow direction promoting enhancement of processes in bead array channels of the microfluidic system of the invention. FIG. 1G demonstrates enhancement of encounters of molecules and detection magnetic beads in in an embodiment in which said magnetic field lines 136 and electric field lines 138 are under an angle of 180°, opposing each other, and causing said magnetic beads 116 to move toward the bottom 101 of said magnetic bead fluidic channel 100 and analyte molecules 134, but not limited to negatively charged nucleic acid or reagents to move toward the positive electrode 122. Positively charged analytes and reagent molecules will move toward negative electrode 124. Fluid flow direction 140 through the magnetic bead detection channel 100 is directed by an external pumping mechanism, not limited to syringe pumping, or pouch dispensing pumping principles operated by the instrument. The present invention uses alternatively an on-cartridge pumping mechanism, with no fluidic connections to the instrument and no mechanical moving parts to direct said fluid flow 140 in said cartridge channels in a desired direction. FIG. 1H demonstrates enhancement of encounters of molecules and detection magnetic beads in the situation in which said magnetic field lines 136 and electric field lines 138 are under an angle of 90°, under a right angle, and the fluid flow 140 in a direction opposing that of the electric field lines 138. Said analyte molecules and various reagents 134 are sequentially brought by fluid flow 140 within the channel where detection is performed on said magnetic beads, enabling reactions on the magnetic beads 116, but not limited to specific binding, hybridization, ligation or other enzymatic or optical reporting reactions such as in situ amplification on beads or real-time reporting using continuous fluorescence monitoring of amplification reaction on said magnetic beads 116. Said magnetic beads 116 can contain pre-bonded reagents such as, but not limited to primers, amplification probes, e.g., circular probes for rolling circle amplification (RCA), which is one of preferred embodiments of the present invention. FIG. 1I demonstrates enhancement of encounters of said analyte or reagent molecules 143 and said detection magnetic beads 116 in an embodiment wherein said magnetic beads 116 are accumulated and focused by magnetic field lines 136 onto electrodes 122 and electric field lines 138 are in parallel, or same in the same direction of magnetic field lines 136. The fluid flow 140, under right angle to both magnetic and electric field affect and stir magnetic beads, causing local convection movement within said magnetic beads 116. The fluid flow 140 is adjusted in such manner that magnetic field 138 keeps magnetic beads 116 focused on the electrodes 122 but causes micro-local convective stirring within the accumulated magnetic beads 116. This combination of convective, magnetic and electrical forces within an extremely small volume on magnetic beads enables further enhancement of biochemical reactions on said magnetic beads and enhances, but not limited to sensitivity of analyte detection, speed of enzymatic reaction such as ligation and polymerization or amplification reactions on said magnetic bead surfaces. FIG. 1J demonstrates enhancement of encounters of said molecules 143 and said detection magnetic beads 116 in an embodiment wherein said magnetic beads 116 are accumulated and focused by magnetic field lines 136 onto top a window of fluidic channel 102 and said electric field lines 138 are under right angle to magnetic field lines 136 and opposite to fluid flow direction 140. Magnetic beads 116 are accumulated during assay protocol on top window 142 of the channel 102. The external magnet can be removed to enable optical detection of signals on magnetic beads 116 accumulated on window 142.

FIG. 1K demonstrates enhancement of encounters of said molecules 143 and said detection magnetic beads 116 in an embodiment wherein said magnetic beads 116 are accumulated by a magnet 108 positioned on top of said fluidic channel 101 and focused by magnetic field lines 136 onto top of said window 142 and said electric field lines 138 are under right angle to magnetic field lines 136 and opposite to said fluid flow direction 140. In this embodiment the electric field is a planar field wherein said positive electrode 122 and said negative electrode 124 are position at said bottom of fluidic channel 101, wherein electric field lines 138 protrude vertically into said channel and through said fluid flow 140. This embodiment enables molecules and reagents 143 to be pushed up vertically in channel 100 through said magnetic beads 116 accumulated on top window 142 of said top of the channel 102 and efficiently mixed with capture or other probes on said magnetic beads to enhance the reactions on said magnetic beads 116 including, but not limited to hybridization, covalent bonding, ligation, polymerization or other biochemical, and not limited to enzymatic reactions. Another embodiment in a similar arrangement as shown in FIG. 1K can have said magnetic beads pre-stored in miniature storages, e.g., indentations at a bottom of said channel 101. The assay protocol starts with bringing an initial solution by said flow 140 that wets said magnetic beads 116 stored, for instance, but not limited as dried beads in said indentations, and magnetizing said beads 116 by said magnet 108 positioned on top of channel 103. Said beads 115 move out vertically from the said storages toward top of window 142 and are accumulated. By moving said magnet 108 along said window 142, said beads 116 can be magnetically moved toward a new desired, different location, where further process can be performed on re-focused beads 116 such as, but not limited to optical detection.

Reference is now made to FIG. 2A which is a schematic illustration of a top view of the housing and microfluidic channels and chambers of the microfluidic device showing components of the electrolytic fluid pumping and bead array chamber of a preferred embodiment of the invention. The microfluidic device comprises at least one or more fluidic channels and chambers, including chamber 200, an electrolytic pump 200 that generates pressure by energizing two electrodes 202 to move the fluid in the channel in a current controlled way, a reagent or magnetic bead storage chamber 206 that has fluidic entrance from the bottom of the chamber, fluidic channels that connect these chambers 204, 208 and 216, the magnetic beads or bead array detection chamber 210, if a detection application is chosen, and the waste chamber 218. The magnetic beads chamber has two parts, with electrodes 210 and 212 in the first part and 214 in the second part. It will be known to those skillful in the art that many other shape designs could be made to focus the beads in the bead array chamber. The electrodes are positioned in the areas where magnetic beads are focused. Several different solutions, but not limited to, sample, sample preparation reagents, analyte amplification and analyte detection can be used and passed through the fluidic lines of the bead array chamber. Multiple other reagent chambers connected to their own electrolytic pumps could be used in parallel channels leading toward the magnetic beads fluidic channels and array.

FIGS. 2B-2F are 3D (three dimensional) illustrations of the structure of manufactured microfluidic device of the invention for implementation of combined magnetic field, electric field and convective enhancement of processes in bead array microfluidic system. FIG. 2B shows single lane bead array, FIG. 2C shows multiple (three) lane array with multiple bead array chambers, FIG. 2D shows multiple (three) lane array with multiple bead array chambers and a focusing magnet in support of localized magnetization over the array, FIG. 2E is a closeup of electrolytic pump, reagent chamber with beads, and FIG. 2F is further close up of the bead array chamber. It is important to note that the fluidics design and arrangement of the microfluidic device of the present invention should be capable to accommodate the magnetic beads chamber and enable magnetic manipulation of magnetic beads and electrical control of ions and analyte and reagent molecules movement within a very small area, preferably 5-8 mm in length of the fluidic channel, more preferably within 3-5 mm.

Reference is now made to FIGS. 3A-3E which are schematic illustrations of the operation steps of the microfluidic device of the invention for implementation of combined magnetic field, electric field and convective enhancement of processes in preferred bead array microfluidic system. The magnetic beads 112 are stored within the device, each type of magnetic bead, e.g., with captures specific to one or more analytes within in a miniature storage chamber preceding the bead array chamber 210. FIG. 3A shows that the beads are fluidically transported under a pressure generated by an electrolysis pump in direction 300 toward the bead array chamber 210. Magnet is brought in proximity of the bottom of the bead array chamber in direction 105. FIG. 3B demonstrates magnetic accumulation of magnetic beads 112 in line with the magnetic field lines 302 toward the narrow bottom end of the chamber to focus and accumulate the magnetic beads. FIG. 3C shows a further step toward combined magnetic field, electric field and convective enhancement of reactions and processes in bead array chambers. The magnet is moved from the bottom of bead array chamber 105 to top of the chamber and magnetic beads are fluidically 304 and 302 dispersed by a pressure and fluid flow from the electrolytic pump, pushing the beads into second part of the bead array chamber. The magnetic beads are now moving toward the top wall of the bead array chamber. An electric field is applied within the second part of the chamber through electrodes 122 and 124, and the analyte molecules, if negatively charge like DNA, RNA and proteins (that can be pre-charged negatively in preceding chambers), moving in the opposite direction 136 from the magnetic beads. This increases accessibility of reagent, analyte and other molecules with the surface of magnetic bead capturing and detection surfaces.

FIG. 3D shows yet another preferred embodiment where the process of electric field, magnetic field control of reactions in the bead chamber shown in FIGS. 3A-3D is further enhanced using one or more vibrating micromotors 310 that are incorporated in the instrument controlling the device and brought into contact 308 with the microfluidics device or cartridge, causing vibrations and enhancing convective transport of molecule around the magnetic bead providing efficient mixing. Typically, micromotors with small load on off-center axes that cause vibration, are operated at high, ultrasonic frequencies in typical range, but not limited to 5,000-20,000 rpm.

Reference is now made to FIGS. 4A-4D which present schematic illustrations of the control of reactions in the microfluidic device of the invention for implementation of combined magnetic field, electric field, and convective processes to enhance different reactions: FIG. 4A: target capturing, and FIGS. 4B-4D show steps of rolling circle isothermal amplification (RCA) of nucleic acid targets on bead array. FIG. 4A shows enhancement of target capturing processes on magnetic beads 400 in the bead array chamber where magnet 105 on top of the chamber attracts magnetic bead upwards 403 and electric field applied through positive and negative electrode 214 moves negatively charged analyte molecules, for instance DNA, RNA or negatively charged proteins 404 toward the positive electrode, in the opposite direction of the magnetic field. This again enhanced contact between magnetic beads containing capture probes, e.g., through biotin-streptavidin binding, but not limited to this chemistry of binding molecules enhances the process of capturing targets from the solution. FIGS. 4B-4D show steps of rolling circle isothermal amplification (RCA) of nucleic acid targets on bead array using enhanced reaction by the magnetic field operating upward in direction 403 and electric field downward in direction 405. The magnetic beads contain all necessary reagents for rolling circle based isothermal amplification of the target, including biotinylated forward primer and reverse primers 409, biotinylated forward primer pre-hybridized to an RCA circular probe. The target 404 is bound to circular probe 406 and upon adding ligase and polymerase, that can be added with or without moving particles magnetically and molecules electrically, the RCA amplification, shown in FIG. 4C occurs anchored or “in situ” on the magnetic beads forming amplicons 410 and 412. In the process, as shown in FIG. 4D some shorter amplicons 414 may wind up in solution surrounding the beads that are re-captured with the counter flow of magnetic beads and electrically driven amplicon in the opposite direction.

Reference is now made to FIGS. 5A-5C that present experimental data for performing an RCA amplification of Hemophilus influenzae DNA target (0.1 uM) on bead array using enhanced using magnetic field movement of the beads. The experimental conditions included: ligation performed using Blunt ligase (NEB) for 5 min at room temperature, RCA amplification using Bst Warm Start Polymerase 2 (NEB), water wash of the beads; all the reagents for RCA were pre-captured on the magnetic beads, and the testing was performed in 200 uL vials. FIG. 5A shows red fluorescence signal photographs obtained after reporting with red reporter and washing excess of reporter. The experiments were performed in the presence and for control in the absence of DNA target. FIG. 5B shows a plot of the fluorescence profile across the target and control samples. FIG. 5C is a graphical presentation of the optical fluorescence and quantitation of the data demonstrating successful RCA amplification on the beads within only 15 minutes of reaction with good specific to non-specific signals ratio >10.2.

Reference is now made to FIGS. 6A-6C that shows repeated experimental data from FIG. 5 for performing RCA amplification of Hemophilus influenzae DNA (0.1 uM) on bead array using enhanced using magnetic field movement of the beads and fluorescence detection on the glass slide after transferring the amplicons from the vials. The experimental conditions included: ligation performed using Blunt ligase (NEB) for 5 min at room temperature, RCA amplification using Bst Warm Start Polymerase 2 (NEB), water wash of the beads; all the reagents for RCA were pre-captured on the magnetic beads, and the testing was performed in 200 uL vials. FIG. 6A shows red fluorescence signal photographs obtained after reporting with red reporter and washing excess of reporter. The experiments were performed in the presence and for control in the absence of DNA target. FIG. 6B shows a plot of the fluorescence profile across the target and control samples. FIG. 6C is a graphical presentation of the optical fluorescence and quantitation of the data demonstrating successful RCA amplification on the beads within only 15 minutes of reaction with good specific to non-specific signals ratio >26.7.

Reference is now made to FIGS. 7A-7C that show experimental data for performing RCA amplification using real-time reporting fluorescence-quencher probe of Acinetobacter baumanii DNA target on bead array (0.1 uM) on bead array using enhanced magnetic field movement of the beads and real-time fluorophore/quencher reporting of the fluorescence detection on the Aluma pressure sensitive adhesive used in the fabrication of the microfluidic chambers of the present invention. The experimental conditions included: ligation performed using Blunt ligase (NEB) for 5 min at room temperature, RCA amplification using Bst Warm Start Polymerase 2 (NEB), water wash of the beads; all the reagents for RCA were pre-captured on the magnetic beads except that the real-time fluorophore/quencher were adding during the RCA amplification, and the testing was performed in 200 uL vials. An exceptionally rapid amplification time of only 2 minutes was obtained using real-time reporting. FIG. 7A shows red fluorescence signal photographs obtained after reporting with real-time reporter and washing excess of reporter. The experiments were performed in the presence and for control in the absence of DNA target. FIG. 7B shows a plot of the fluorescence profile across the target and control samples. FIG. 7C is a graphical presentation of the optical fluorescence and quantitation of the data demonstrating successful RCA amplification on the beads within only 2 minutes of reaction with good specific to non-specific signals ratio >9.3.

Reference is now made to FIGS. 8A-8C that show experimental data demonstrating a comparison of enhancement of the target capture process in in conditions: combined electric+magnetic field vs. magnetic field only applied to target capture using a Acinetobacter baumanii oligo DNA target fluorescently labeled and biotinylated to compare the capturing efficiency in those conditions. and performing a comparison between the fluorescence signals obtained. FIG. 8A shows red fluorescence signal photographs obtained after reporting the capturing of the 10 nM target where the process is enhanced: (i) by applying electric field movement of molecules (at 0.5 mA/1 min applied across the electrodes in the bead chamber combined with the magnetic movement of the beads in the opposite direction, and (ii) same reaction by applying magnetic movement of magnetic beads capturing the target in solution with no electric field applied. FIG. 8B shows a plot of the fluorescence profile across the target capture when magnetic+electric field was applied and a control when only magnetic field was applied. FIG. 8C is a graphical presentation of the optical fluorescence and quantitation of the data clearly demonstrating much more efficient transport by combining electric/magnetic enhancement of the reactions, with the specific to non-specific signals ratio >8.5, and magnetic enhancement only, with the specific to non-specific signals ratio >8.5, respectively.

Reference is now made to FIGS. 9A-9D which show photographs of a manufactured microfluidic device with steps operating the microfluidic device of the invention for implementation of combined magnetic field, electric field and convective enhancement of processes in bead array microfluidic system using fluorescently labeled beads to confirm the operation of each step in the device. Accumulation of the magnetic beads and extraction from the fluid at the bottom of the bead array chambers shown in FIG. 9A to FIG. 9B, and is clearly visible in the narrow portions of the bead array chamber envisioned for focusing of each bead in their bead chamber. By moving the magnet to the top of the bead array chamber, a dispersion of the beads toward the other focusing portion of the bead array chamber is visible in FIGS. 9C and 9D, and the effect of the electric field applied did not affect the movement of the beads, but the charged molecules in the chamber (not visible through the fluorescence data).

Reference is now made to FIGS. 10A-10C which show assay protocols and data for detection and identification of Escherichia coli pathogen in whole blood clinical samples on a manufactured microfluidic device with magnetic bead array operated under combined magnetic field, electric field and convective enhancement of processes in bead array microfluidic system. FIG. 10A is a photograph of the microfluidic device with an electrolytic chamber housing stainless steel sacrificial electrodes for generating hydrogen and oxygen needed for pumping reagents toward the microfluidic channel containing the magnetic bead array. The microfluidic system shown in FIG. 10A-10C is a combination of system operation shown in FIG. 11, where the magnetic beads are disposed onto electrodes and kept focused under magnetic field. The electric field is implemented as shown in FIG. 1K, where stainless steel electrodes, circular working electrodes with beads, and counter electrodes of same stainless steel material are configured as a planar array. The streptavidin coated magnetic beads contained biotinylated forward primer, hybridized to circular RCA probes specific to E. coli, as well as forward and reverse primers. The assay followed the following fluidics protocol in providing fluids on the magnetic bead array: (i) Target E. coli sample (10⁴ CFU/mL was brought by the fluid flow over the magnetic bead array and an electric field was applied for 30 s under constant current of 1.5 mA/7.0 V; (ii) The ligation solution (Blunt/T4 DNA ligase, NEB) was added to the array, 3 min at room temperature; (iii) An RCA solution containing Bst polylmerase, Warm Start (NEB) and real time red reporter Quencher/Fluorophor reagent was applied over the magnetic bead array for 3 min at 65 C; (iv) post-addressing of RCA amplicon was performed under an applied electric field of 0.5 mA/3.0 V, 30 s. FIG. 10B shows fluorescent data obtained on the magnetic bead array wherein assay performance was enhanced by combined magnetic and electric field applied on the array. A schematic insert over the fluorescent image shows how E. coli specific RCA circular probes (Cp) and non-specific probe were applied and positioned on the magnetic bead array. FIG. 10C shows red fluorescence signal photographs obtained after only 3 minutes of isothermal RCA amplification accelerated under electric/magnetic field on the magnetic bead array. FIG. 10C shows a plot of quantification of the fluorescence signals on the magnetic beads across the bead array when magnetic+electric field was applied demonstrating rapid and specific detection and identification of Escherichia coli targets with satisfactory specific to non-specific signals ratio >3.9.

FIG. 11 is a block diagram illustrating an example wired or wireless system 550 that may be used in connection with various embodiments described herein. For example, the system 550 may be used as or in conjunction with controlling the operation of the microfluidic system as described herein. The system 550 can be a conventional personal computer, computer server, personal digital assistant, smart phone, tablet computer, or any other processor enabled device that is capable of wired or wireless data communication. Other computer systems and/or architectures may be also used, as will be clear to those skilled in the art.

The system 550 preferably includes one or more processors, such as processor 560. Additional processors may be provided, such as an auxiliary processor to manage input/output, an auxiliary processor to perform floating point mathematical operations, a special-purpose microprocessor having an architecture suitable for fast execution of signal processing algorithms (e.g., digital signal processor), a slave processor subordinate to the main processing system (e.g., back-end processor), an additional microprocessor or controller for dual or multiple processor systems, or a coprocessor. Such auxiliary processors may be discrete processors or may be integrated with the processor 560.

The processor 560 is preferably connected to a communication bus 555. The communication bus 555 may include a data channel for facilitating information transfer between storage and other peripheral components of the system 550. The communication bus 555 further may provide a set of signals used for communication with the processor 560, including a data bus, address bus, and control bus (not shown). The communication bus 555 may comprise any standard or non-standard bus architecture such as, for example, bus architectures compliant with industry standard architecture (“ISA”), extended industry standard architecture (“EISA”), Micro Channel Architecture (“MCA”), peripheral component interconnect (“PCI”) local bus, or standards promulgated by the Institute of Electrical and Electronics Engineers (“IEEE”) including IEEE 488 general-purpose interface bus (“GPM”), IEEE 696/S-100, and the like.

System 550 preferably includes a main memory 565 and may also include a secondary memory 570. The main memory 565 provides storage of instructions and data for programs executing on the processor 560. The main memory 565 is typically semiconductor-based memory such as dynamic random access memory (“DRAM”) and/or static random access memory (“SRAM”). Other semiconductor-based memory types include, for example, synchronous dynamic random access memory (“SDRAM”), Rambus dynamic random access memory (“RDRAM”), ferroelectric random access memory (“FRAM”), and the like, including read only memory (“ROM”).

The secondary memory 570 may optionally include an internal memory 575 and/or a removable medium 580, for example a floppy disk drive, a magnetic tape drive, a compact disc (“CD”) drive, a digital versatile disc (“DVD”) drive, etc. The removable medium 580 is read from and/or written to in a well-known manner. Removable storage medium 580 may be, for example, a floppy disk, magnetic tape, CD, DVD, SD card, etc.

The removable storage medium 580 is a non-transitory computer readable medium having stored thereon computer executable code (i.e., software) and/or data. The computer software or data stored on the removable storage medium 580 is read into the system 550 for execution by the processor 560.

In alternative embodiments, secondary memory 570 may include other similar means for allowing computer programs or other data or instructions to be loaded into the system 550. Such means may include, for example, an external storage medium 595 and an interface 570. Examples of external storage medium 595 may include an external hard disk drive or an external optical drive, or and external magneto-optical drive.

Other examples of secondary memory 570 may include semiconductor-based memory such as programmable read-only memory (“PROM”), erasable programmable read-only memory (“EPROM”), electrically erasable read-only memory (“EEPROM”), or flash memory (block oriented memory similar to EEPROM). Also included are any other removable storage media 580 and communication interface 590, which allow software and data to be transferred from an external medium 595 to the system 550.

System 550 may also include an input/output (“I/O”) interface 585. The I/O interface 585 facilitates input from and output to external devices. For example the I/O interface 585 may receive input from a keyboard or mouse and may provide output to a display 587. The I/O interface 585 is capable of facilitating input from and output to various alternative types of human interface and machine interface devices alike.

System 550 may also include a communication interface 590. The communication interface 590 allows software and data to be transferred between system 550 and external devices (e.g. printers), networks, or information sources. For example, computer software or executable code may be transferred to system 550 from a network server via communication interface 590. Examples of communication interface 590 include a modem, a network interface card (“NIC”), a wireless data card, a communications port, a PCMCIA slot and card, an infrared interface, and an IEEE 1394 fire-wire, just to name a few.

Communication interface 590 preferably implements industry promulgated protocol standards, such as Ethernet IEEE 802 standards, Fiber Channel, digital subscriber line (“DSL”), asynchronous digital subscriber line (“ADSL”), frame relay, asynchronous transfer mode (“ATM”), integrated digital services network (“ISDN”), personal communications services (“PCS”), transmission control protocol/Internet protocol (“TCP/IP”), serial line Internet protocol/point to point protocol (“SLIP/PPP”), and so on, but may also implement customized or non-standard interface protocols as well.

Software and data transferred via communication interface 590 are generally in the form of electrical communication signals 605. These signals 605 are preferably provided to communication interface 590 via a communication channel 600. In one embodiment, the communication channel 600 may be a wired or wireless network, or any variety of other communication links. Communication channel 600 carries signals 605 and can be implemented using a variety of wired or wireless communication means including wire or cable, fiber optics, conventional phone line, cellular phone link, wireless data communication link, radio frequency (“RF”) link, or infrared link, just to name a few.

Computer executable code (i.e., computer programs or software) is stored when executed, enable the system 550 to perform the various functions of the present invention as previously described.

In this description, the term “computer readable medium” is used to refer to any non-transitory computer readable storage media used to provide computer executable code (e.g., software and computer programs) to the system 550. Examples of these media include main memory 565, secondary memory 570 (including internal memory 575, removable medium 580, and external storage medium 595), and any peripheral device communicatively coupled with communication interface 590 (including a network information server or other network device). These non-transitory computer readable mediums are means for providing executable code, programming instructions, and software to the system 550.

In an embodiment that is implemented using software, the software may be stored on a computer readable medium and loaded into the system 550 by way of removable medium 580, I/O interface 585, or communication interface 590. In such an embodiment, the software is loaded into the system 550 in the form of electrical communication signals 605. The software, when executed by the processor 560, preferably causes the processor 560 to perform the inventive features and functions previously described herein.

The system 550 also includes optional wireless communication components that facilitate wireless communication over a voice and over a data network (or otherwise described herein). The wireless communication components comprise an antenna system 610, a radio system 615 and a baseband system 620. In the system 550, radio frequency (“RF”) signals are transmitted and received over the air by the antenna system 610 under the management of the radio system 615.

In one embodiment, the antenna system 610 may comprise one or more antennae and one or more multiplexors (not shown) that perform a switching function to provide the antenna system 610 with transmit and receive signal paths. In the receive path, received RF signals can be coupled from a multiplexor to a low noise amplifier (not shown) that amplifies the received RF signal and sends the amplified signal to the radio system 615.

In alternative embodiments, the radio system 615 may comprise one or more radios that are configured to communicate over various frequencies. In one embodiment, the radio system 615 may combine a demodulator (not shown) and modulator (not shown) in one integrated circuit (“IC”). The demodulator and modulator can also be separate components. In the incoming path, the demodulator strips away the RF carrier signal leaving a baseband receive audio signal, which is sent from the radio system 615 to the baseband system 620.

If the received signal contains audio information, then baseband system 620 decodes the signal and converts it to an analog signal. Then the signal is amplified and sent to a speaker. The baseband system 620 also receives analog audio signals from a microphone. These analog audio signals are converted to digital signals and encoded by the baseband system 620. The baseband system 620 also codes the digital signals for transmission and generates a baseband transmit audio signal that is routed to the modulator portion of the radio system 615. The modulator mixes the baseband transmit audio signal with an RF carrier signal generating an RF transmit signal that is routed to the antenna system and may pass through a power amplifier (not shown). The power amplifier amplifies the RF transmit signal and routes it to the antenna system 610 where the signal is switched to the antenna port for transmission.

The baseband system 620 is also communicatively coupled with the processor 560. The central processing unit 560 has access to data storage areas 565 and 570. The central processing unit 560 is preferably configured to execute instructions (i.e., computer programs or software) that can be stored in the memory 565 or the secondary memory 570. Computer programs can also be received from the baseband processor 610 and stored in the data storage area 565 or in secondary memory 570, or executed upon receipt. Such computer programs, when executed, enable the system 550 to perform the various functions of the present invention as previously described. For example, data storage areas 565 may include various software modules (not shown) that are executable by processor 560.

Various embodiments may also be implemented primarily in hardware using, for example, components such as application specific integrated circuits (“ASICs”), or field programmable gate arrays (“FPGAs”). Implementation of a hardware state machine capable of performing the functions described herein will also be apparent to those skilled in the relevant art. Various embodiments may also be implemented using a combination of both hardware and software.

Furthermore, those of skill in the art will appreciate that the various illustrative logical blocks, modules, circuits, and method steps described in connection with the above described figures and the embodiments disclosed herein can often be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled persons can implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the invention. In addition, the grouping of functions within a module, block, circuit or step is for ease of description. Specific functions or steps can be moved from one module, block or circuit to another without departing from the invention.

Moreover, the various illustrative logical blocks, modules, and methods described in connection with the embodiments disclosed herein can be implemented or performed with a general purpose processor, a digital signal processor (“DSP”), an ASIC, FPGA or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general-purpose processor can be a microprocessor, but in the alternative, the processor can be any processor, controller, microcontroller, or state machine. A processor can also be implemented as a combination of computing devices, for example, a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

Additionally, the steps of a method or algorithm described in connection with the embodiments disclosed herein can be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. A software module can reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium including a network storage medium. An exemplary storage medium can be coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium can be integral to the processor. The processor and the storage medium can also reside in an ASIC.

In other embodiments, a method is provided for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising:

-   -   a) providing a microfluidic system comprising a microfluidic         device, wherein the microfluidic device comprises:         -   i) a housing, wherein the housing comprises a top end and a             bottom end;ii) a plurality of reagent chambers and a             plurality of pressure-generating chambers, wherein the             reagent chambers and the pressure-generating chambers are             positioned in the housing, and wherein:             -   aa) the pressure-generating chambers produce a                 pressure-generating fluid using no mechanical moving                 parts;             -   bb) the reagent chambers are connected by at least one                 gas channel at the top end of the housing to at least                 one of the pressure-generating chambers;             -   cc) the reagent chambers are connected by one or more                 liquid channels at the bottom end of the housing to one                 or more of the pressure-generating chambers; and             -   dd) the reagent chambers comprise one or more reagent                 fluids comprising one or more sets of magnetic beads                 comprising capture probes specific to the one or more                 target analytes;         -   iii) a top substrate enclosing the pressure-generating fluid             chambers, wherein the top substrate comprises fluidic             channels connecting the pressure-generating chambers to one             or more vent holes, thereby enabling movement of one or more             reagent fluids in the one or more liquid channels at the             bottom end of the housing; and         -   iv) a bottom substrate enclosing the reagent chambers;         -   wherein the microfluidic system is configured to achieve             passive flow resistance during filling of the microfluidic             device with the pressure-generating fluid to prevent mixing             of the pressure-generating fluid with the reagents when the             microfluidic system is not in operation;     -   b) activating the one or more pressure-generating chambers to         pump the pressure-generating fluid toward the one or more         reagent chambers and controlling and balancing pressure of the         pressure-generating fluid to achieve active flow resistance         resulting in the movement of the one or more reagent fluids in a         desired direction, whereby fluidic movement of the one or more         sets of magnetic beads is enabled and controlled; and     -   c) combining the one or more sets of magnetic beads with one or         more sample solutions, analyte detection solutions, and/or wash         solutions in a localized area of one of the microfluidic         channels, and magnetically releasing, dispersing, focusing, and         recapturing the one or more sets of magnetic beads, thereby         enhancing magnetic bead-based detection of the one or more         target analytes.

In some embodiments, the method for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels further comprises achieving passive flow resistance during filling of the microfluidic device, further comprising the steps of:

-   -   ai) filling the plurality of pressure-generating chambers with         pressure-generating fluid;     -   bi) enclosing the housing and the plurality of         pressure-generating fluid chambers, the gas channels, and the         plurality of reagent chambers with the top substrate such that         the fluidic channels make desired connections between the         chambers and vent holes enabling movement of one or more reagent         fluids in liquid channels at the bottom end of the housing; and     -   ci) inverting the microfluidic device and filling the plurality         of reagent chambers with the one or more reagent fluids and         enclosing the reagent chambers, the one or more reagent fluids,         and the liquid channels with a bottom substrate at the bottom         end of the housing.

In some embodiments, the method is executed by an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.

In some embodiments of the method, the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.

In some embodiments of the method, the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.

In some embodiments of the method, the one or more reagent fluids comprise one or more reagents for extraction, amplification, or detection, comprising one or more biomarkers, nutrients, and/or chemicals.

In some embodiments of the method, the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.

In some embodiments of the method, the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.

In some embodiments of the method, electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.

In some embodiments of the method, the microfluidic device further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.

In some embodiments of the method, the microfluidic system is configured to enable the gas produced by electrolysis to control pH and/or conductivity reactions in the one or more of the plurality of reagent chambers.

In some embodiments of the method, the microfluidic system further comprises one or more gas permeable membranes atop the plurality of pressure generation chambers, wherein the one or more gas permeable membranes separate liquid and gas pressure-generating fluids in the pressure-generating chambers while allowing permeation of pressure-generating fluid into the fluidic channels without mixing between the pressure-generating fluid and the one or more reagent fluids in the plurality of reagent chambers.

In some embodiments of the method, the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.

In some embodiments of the method, the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.

In some embodiments of the method, the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.

In some embodiments of the method, the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes.

In some embodiments of the method, the microfluidic system further comprises vibrating micromotors configured to enhance convective transport and mixing.

General Definitions

Terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term “including” should be read as mean “including, without limitation” or the like; the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and adjectives such as “conventional,” “traditional,” “standard,” “known” and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass conventional, traditional, normal, or standard technologies that may be available or known now or at any time in the future. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/or” unless expressly stated otherwise. Furthermore, although item, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the subject matter of the present invention. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments±100%, in some embodiments±50%, in some embodiments±20%, in some embodiments±10%, in some embodiments±5%, in some embodiments±1%, in some embodiments±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

As used herein, the terms “amplify,” “amplification,” “nucleic acid amplification,” or the like, refer to the production of multiple copies of a nucleic acid template (e.g., a template DNA molecule), or the production of multiple nucleic acid sequence copies that are complementary to the nucleic acid template (e.g., a template DNA molecule). As used herein, the term “magnetic bead,” means any bead or particle that is magnetically responsive. Magnetically responsive material may constitute substantially all of a bead, a portion of a bead, or only one component of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent.

As used herein, the term “biomarker” refers to any gene, RNA, or protein, for example a gene, RNA, or protein whose level of expression in a cell or tissue is altered in some way compared to that of a normal or healthy cell or tissue. In some embodiments, the amount of biomarker may be changed. In other embodiments, the biomarker may be differentially modified in some way.

As used herein, the term “level of expression” of a biomarker refers to the amount of biomarker detected. Levels of biomarker can be detected at the transcriptional level, the translational level, and the post-translational level, for example. “mRNA expression levels” refers to the amount of mRNA detected in a sample and “protein expression levels” refers to the amount of protein detected in a sample.

As used herein, the term “array” or “microarray” refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

As used herein, the term “nucleic acid” or “polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

As used herein, the term “oligonucleotide” refers to a relatively short polynucleotide. This includes, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs.

As used herein, the term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.

As used herein, the term “probe” denotes a defined nucleic acid segment which can be used to identify a specific polynucleotide sequence present in samples, wherein the nucleic acid segment comprises a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.

As used herein, the terms “complementary” or “complement thereof” refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. For the purpose of the presently disclosed subject matter, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. “Complement” is used herein as a synonym from “complementary polynucleotide,” “complementary nucleic acid” and “complementary nucleotide sequence”. These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

The terms “patient,” “individual,” or “subject” are used interchangeably herein, and refer to a mammal, particularly, a human. A “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. The patient may have mild, intermediate or severe disease. The patient may be treatment naive, responding to any form of treatment, or refractory. The patient may be an individual in need of treatment or in need of diagnosis based on particular symptoms or family history. In some cases, the terms may refer to treatment in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.

The terms “sample,” “patient sample,” “biological sample,” and the like, encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic or monitoring assay. The patient sample may be obtained from a healthy subject or a diseased patient. Moreover, a sample obtained from a patient can be divided and only a portion may be used for diagnosis. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis. The definition specifically encompasses blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, cerebrospinal fluid, urine, saliva, stool and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof In a specific embodiment, a sample comprises a blood sample. In another embodiment, a serum sample is used. The definition also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations. The terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry.

A “suitable control,” “appropriate control” or a “control sample” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, and the like, determined in a cell, organ, or patient, e.g., a control or normal cell, organ, or patient, exhibiting, for example, normal traits.

All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims. 

1. A microfluidic system for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising: a) a microfluidic device comprising a housing, wherein the housing comprises a top end and a bottom end; b) a plurality of reagent chambers and a plurality of pressure-generating chambers, wherein the reagent chambers and the pressure-generating chambers are positioned in the housing, and wherein: i) the pressure-generating chambers produce a pressure-generating fluid using no mechanical moving parts; ii) the reagent chambers are connected by at least one gas channel at the top end of the housing to at least one of the pressure-generating chambers; iii) the reagent chambers are connected by one or more liquid channels at the bottom end of the housing to one or more of the pressure-generating chambers; and iv) the reagent chambers comprise one or more reagent fluids comprising one or more sets of magnetic beads comprising capture probes specific to the one or more target analytes; c) a top substrate enclosing the pressure-generating fluid chambers, wherein the top substrate comprises fluidic channels connecting the pressure-generating chambers to one or more vent holes, thereby enabling movement of the one or more reagent fluids in the one or more liquid channels at the bottom end of the housing; and d) a bottom substrate enclosing the reagent chambers; wherein the microfluidic system is configured to achieve passive flow resistance during filling of the microfluidic device with the pressure-generating fluid to prevent mixing of the pressure-generating fluid with the reagents when the microfluidic system is not in operation; wherein the microfluidic system is configured such that the movement of the one or more reagent fluids is enabled by activating the one or more pressure-generating chambers to pump the pressure-generating fluid toward the one or more reagent chambers and controlling and balancing pressure of the pressure-generating fluid to achieve active flow resistance resulting in the movement of the one or more reagent fluids in a desired direction, whereby fluidic movement of the one or more sets of magnetic beads is enabled and controlled; and wherein the microfluidic system is configured to enable combining the one or more sets of magnetic beads with one or more sample solutions, analyte detection solutions, and/or wash solutions in a localized area of one of the microfluidic channels, whereby the microfluidic system is configured to magnetically release, disperse, focus, and recapture the one or more sets of magnetic beads, thereby enhancing magnetic bead-based detection of the one or more target analytes.
 2. The microfluidic system of claim 1, wherein achieving passive flow resistance during filling of the microfluidic device comprises the steps of: aa) filling the plurality of pressure-generating chambers with pressure-generating fluid; bb) enclosing the housing and the plurality of pressure-generating fluid chambers, the gas channels, and the plurality of reagent chambers with the top substrate such that the fluidic channels make desired connections between the chambers and vent holes enabling movement of one or more reagent fluids in liquid channels at the bottom end of the housing; and cc) inverting the microfluidic device and filling the plurality of reagent chambers with the one or more reagent fluids and enclosing the reagent chambers, the one or more reagent fluids, and the liquid channels with a bottom substrate at the bottom end of the housing.
 3. The microfluidic system of claim 2, further comprising an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.
 4. The microfluidic system of claim 3, wherein the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.
 5. The microfluidic system of claim 4, wherein the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.
 6. The microfluidic system of claim 5, wherein the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.
 7. The microfluidic system of claim 6, wherein the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.
 8. The microfluidic system of claim 7, wherein electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.
 9. The microfluidic system of claim 8, further comprising one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.
 10. The microfluidic system of claim 9, configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.
 11. The microfluidic system of claim 10, configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.
 12. The microfluidic system of claim 11, configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.
 13. The microfluidic system of claim 11, configured to enable focusing magnetic beads atop the one or more electrodes.
 14. The microfluidic system of claim 13, further comprising vibrating micromotors configured to enhance convective transport and mixing.
 15. A method for implementing combined magnetic field, electric field, and convective enhancement of magnetic bead-based detection of one or more target analytes based on active control of flow resistance in microfluidic channels, comprising: a) providing a microfluidic system comprising a microfluidic device, wherein the microfluidic device comprises: i) a housing, wherein the housing comprises a top end and a bottom end; ii) a plurality of reagent chambers and a plurality of pressure-generating chambers, wherein the reagent chambers and the pressure-generating chambers are positioned in the housing, and wherein: aa) the pressure-generating chambers produce a pressure-generating fluid using no mechanical moving parts; bb) the reagent chambers are connected by at least one gas channel at the top end of the housing to at least one of the pressure-generating chambers; cc) the reagent chambers are connected by one or more liquid channels at the bottom end of the housing to one or more of the pressure-generating chambers; and dd) the reagent chambers comprise one or more reagent fluids comprising one or more sets of magnetic beads comprising capture probes specific to the one or more target analytes; iii) a top substrate enclosing the pressure-generating fluid chambers, wherein the top substrate comprises fluidic channels connecting the pressure-generating chambers to one or more vent holes, thereby enabling movement of one or more reagent fluids in the one or more liquid channels at the bottom end of the housing; and iv) a bottom substrate enclosing the reagent chambers; wherein the microfluidic system is configured to achieve passive flow resistance during filling of the microfluidic device with the pressure-generating fluid to prevent mixing of the pressure-generating fluid with the reagents when the microfluidic system is not in operation; b) activating the one or more pressure-generating chambers to pump the pressure-generating fluid toward the one or more reagent chambers and controlling and balancing pressure of the pressure-generating fluid to achieve active flow resistance resulting in the movement of the one or more reagent fluids in a desired direction, whereby fluidic movement of the one or more sets of magnetic beads is enabled and controlled; and c) combining the one or more sets of magnetic beads with one or more sample solutions, analyte detection solutions, and/or wash solutions in a localized area of one of the microfluidic channels, and magnetically releasing, dispersing, focusing, and recapturing the one or more sets of magnetic beads, thereby enhancing magnetic bead-based detection of the one or more target analytes.
 16. The method of claim 15, comprising achieving passive flow resistance during filling of the microfluidic device, further comprising the steps of: ai) filling the plurality of pressure-generating chambers with pressure-generating fluid; bi) enclosing the housing and the plurality of pressure-generating fluid chambers, the gas channels, and the plurality of reagent chambers with the top substrate such that the fluidic channels make desired connections between the chambers and vent holes enabling movement of one or more reagent fluids in liquid channels at the bottom end of the housing; and ci) inverting the microfluidic device and filling the plurality of reagent chambers with the one or more reagent fluids and enclosing the reagent chambers, the one or more reagent fluids, and the liquid channels with a bottom substrate at the bottom end of the housing.
 17. The method of claim 16, wherein the method is executed by an automated electronics interface and software control configured to control and balance the pressure of the pressure-generating fluid, wherein the automated electronics interface and software control is programmed to execute a reproducible protocol for operation of the microfluidic device.
 18. The method of claim 17, wherein the pressure of the pressure-generating fluid in the plurality of pressure-generating chambers is generated using electrolytic gas evolution, thermal heating, catalytic heating, ultrasonic means, electrophoretic means, or dielectrophoretic means.
 19. The method of claim 18, wherein the microfluidic device is configured to control the pressure of the pressure-generating fluid electronically using electrodes, electronic contacts, and/or switches embedded in the housing.
 20. The method of claim 19, wherein the one or more reagent fluids comprise one or more reagents for extraction, amplification, or detection, comprising one or more biomarkers, nutrients, and/or chemicals.
 21. The method of claim 20, wherein the one or more pressure-generating fluids comprise aqueous or non-aqueous liquids.
 22. The method of claim 21, wherein the one or more vent-holes are embedded within the top substrate of the housing atop one or more pressure-generating chambers or one or more reagent chambers.
 23. The method of claim 22, wherein electrolytic gas evolution generates the pressure of the pressure-generating fluid by electrolysis of the pressure-generating fluid, wherein the pressure-generating fluid comprises water, an inorganic salt solution, or a conductive organic solution, and wherein electrolysis of the pressure-generating fluid produces a gas comprising oxygen, hydrogen, and/or chlorine.
 24. The method of claim 23, wherein the microfluidic device further comprises one or more electrodes for electrolytic gas evolution, wherein the one or more electrodes comprise anodic corrosion-stable noble metal electrodes or one or more anodically sacrificial electrodes, wherein the one or more anodically sacrificial electrodes comprise stainless steel, aluminum, copper, carbon, carbon inks, plated electrodes, and/or screen-printed electrodes.
 25. The method of claim 24, wherein the microfluidic system is configured to enable the gas produced by electrolysis to control pH and/or conductivity reactions in the one or more of the plurality of reagent chambers.
 26. The method of claim 25, wherein the microfluidic system further comprises one or more gas permeable membranes atop the plurality of pressure generation chambers, wherein the one or more gas permeable membranes separate liquid and gas pressure-generating fluids in the pressure-generating chambers while allowing permeation of pressure-generating fluid into the fluidic channels without mixing between the pressure-generating fluid and the one or more reagent fluids in the plurality of reagent chambers.
 27. The method of claim 26, wherein the microfluidic system is configured to pump the pressure-generating fluid toward one of the plurality of reagent chambers that comprises one of the vent holes, or wherein the pressure-generating fluid is pumped toward one of the plurality of pressure generation chambers that comprises a vent hole, thereby causing a high flow velocity and generating a Venturi vacuum, wherein the Venturi vacuum enables control of fluid flow resistance and/or fluid flow velocity.
 28. The method of claim 27, wherein the microfluidic system is configured to enable one or more magnets to generate one or more magnetic fields and one or more electrodes to generate one or more electric fields, wherein the one or more magnetic fields and the one or more electric fields enable movement of the one or more sets of magnetic beads relative to movement of the one or more analytes.
 29. The method of claim 29, wherein the microfluidic system is configured to enable movement of the one or more magnetic beads and the one or more analytes in the same direction or in different directions, wherein the different directions comprise opposite directions.
 30. The method of claim 29, wherein the microfluidic system is configured to enable focusing magnetic beads atop the one or more electrodes.
 31. The method of claim 30, further comprising vibrating micromotors configured to enhance convective transport and mixing. 